Retinoblastoma occurs in two different genetic forms, hereditary and nonhereditary. These forms can often be distinguished on the basis of clinical features, family history, pedigree analysis (linkage to a polymorphic marker enzyme esterase D) and chromosome analysis. Accumulated evidence strongly favors the existence of a single retinoblastoma susceptibility locus for both types of retinoblastoma. Tumorigenesis appears to involve alterations in both alleles at this locus. In hereditary retinoblastoma, one defective or missing allele is inherited, and expression of the tumor depends on additional mutation or deletion of the other allele. We propose to isolate the retinoblastoma susceptibility (Rb) gene by "chromosome walking" in both directions from the esterase D gene, which is tightly linked to Rb in band q14.2 of chromosome 13. A clone of the esterase D gene is available in our laboratory. The best candidates for the Rb gene will be located in DNA segments mapping to band 13q14 which hybridize with mRNA from normal human embryonic retinal (HER) cells but not with mRNA from retinoblastoma cells. Alternatively, mRNA from retinoblastoma cells will be used to "subtract out" cDNA of HER cells and the remaining cDNA clones will be mapped back to the 13q14 region. To positively identify the Rb gene, candidate clones can be introduced into retinoblastoma cells while observing for phenotypic reversion of tumor cells. An alternative method is to introduce the antisense strands of these genes into normal HER cells and observe for transformation to cells with features of retinoblastoma. Protein(s) encoded by the Rb gene will be deduced from the nucleotide sequence and identified by immunoprecipitation with antibodies to synthetic peptides of the corresponding protein(s). Isolation of the Rb gene is a crucial step in determining the molecular oncogenesis of retinoblastoma. In addition, this gene can be used as a probe in a restriction fragment length polymorphism assay to diagnose hereditary versus non-hereditary disease, both pre- and post-natally.